The present invention is directed to the protection and enhancement of cells, tissues and organs of a mammalian body that may be isolated or separated from the vasculature by an endothelial cell barrier. Protection is provided to cells, tissues and organs in situ as well as those that may be temporarily removed, for instance, for transplant. Protection and enhancement is offered to cells, tissues and organs that are erythropoietin responsive or those that depend on erythropoietin-responsive cells for at least an aspect of function or viability.
For many years, the only clear physiological role of erythropoietin (EPO) had been its control of the production of red blood cells. Recently, several lines of evidence suggest that EPO, as a member of the cytokine superfamily, performs other important physiologic functions which are mediated through interaction with the erythropoietin receptor (EPO-R). These actions include mitogenesis, modulation of calcium influx into smooth muscle cells and neural cells, and effects on intermediary metabolism. It is believed that EPO provides compensatory responses that serve to improve hypoxic cellular microenvironments. Although studies have established that EPO injected intracranially protects neurons against hypoxic neuronal injury, intracranial administration is an impractical and unacceptable route of administration for therapeutic use, particularly for normal individuals. Furthermore, previous studies of anemic patients given EPO have concluded that peripherally-administered EPO is not transported into the brain (Marti et al., 1997, Kidney Int. 51:416-8). Copending U.S. application Ser. No. 09/547,220, the entire contents of which is incorporated herein by reference in its entirety, describes the heretofore unrecognized and unappreciated property that a peripherally-administered erythropoietin is capable of crossing an endothelial cell barrier (e.g., the blood-brain barrier) and providing protection and enhancement of excitable tissue across that barrier, for example, for protection or enhancement of neuronal function. It also describes the use of an erythropoietin as a carrier molecule to facilitate the penetration across an endothelial cell barrier, such as the blood-brain barrier, to serve as a carrier for compounds which alone normally cannot cross such barriers.
It is towards the use of an erythropoietin for protecting or enhancing erythropoietin-responsive cells and associated cells, tissues and organs in situ and ex vivo, as well as delivery of an erythropoietin across an endothelial cell barrier for the purpose of protecting and enhancing erythropoietin-responsive cells and associated cells, tissues and organs distal to the vasculature, that the present invention is directed.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.
In its broadest aspect, the present invention is directed to compositions and methods for protecting, maintaining or enhancing the function or viability of an erythropoietin-responsive mammalian cell and associated cells, tissues and organs, using compounds which may or may not have erythropoietic activity.
In one aspect, the invention is directed to protecting, maintaining or enhancing the viability of an erythropoietin-responsive mammalian cell, and associated cells, tissues and organs, wherein the mammalian cell, tissue or organ is distal to the vasculature by virtue of a tight endothelial cell barrier. The method involves administering to the vasculature an amount of an erythropoietin which is capable of translocation across the intact endothelial cell barrier and providing benefits to erythropoietin-responsive cells and to associated cells, tissues and organs dependent thereon distal to the vasculature.
The invention is based, in part, on the Applicant""s discovery that particularly for chronic treatment for the various purposes herein, it is desirable to utilize an erythropoietin with positive effects on erythropoietin-responsive cells and tissues but lacking erythropoietic activity, to prevent an undesirable rise in hemoglobin concentrations and hematocrit over the course of therapy. Such non-erythropoietic forms of EPO may exhibit lack of stimulating erythropoiesis by any of several non-limiting reasons, such as altered pharmacokinetics or altered activity. For example, a suitable non-erythropoietic form of erythropoietin may have too short of a half-life in circulation to be present at a concentration adequate to recruit proerythroblasts via the erythropoietin receptor. Alternatively, a chemically-modified, mutant, or otherwise altered form of erythropoietin which is not recognized by the erythropoietin receptor on proerythroblasts but maintains or has improved activity at enhancing viability and other positive effects on erythropoietin-responsive cells is embraced herein. As described herein, in the case wherein the EPO is non-erythropoietic, the range of doses may be as described, or may be lower depending on the potency of the non-erythropoietic molecule. Units of activity may be determined based on equivalent activity against the target cells and tissues described herein, as the Units normally used for EPO relate to erythropoiesis and are irrelevant to non-erythropoietic EPOs.
The invention is also based, in part, on the Applicant""s discovery that organs and other bodily parts isolated from a mammalian body, such as those intended for transplant, are benefitted by exposure to an erythropoietin.
Thus, one aspect of the invention is directed to a method for protecting, maintaining or enhancing the viability or function of a mammalian cell, tissue, organ or bodily part which includes an erythropoietin-responsive cell or tissue, in which the cell, tissue, organ or bodily part is isolated from the mammalian body. The method includes at least exposing the isolated mammalian cell, tissue, organ or bodily part to an amount of an erythropoietin for a duration which is effective to protect, maintain or enhance the aforementioned viability or function. In non-limiting examples, isolated refers to restricting or clamping the vasculature of or to the cell, tissue, organ or bodily part, such as may be performed during surgery; bypassing the vasculature of the cell, tissue, organ or bodily part; removing the cell, tissue, organ or bodily part from the mammalian body, such may be done in advance of xenotransplantation or prior to and during autotransplantation; or traumatic amputation of a cell, tissue, organ or bodily part. Thus, this aspect of the invention pertains both to the perfusion with an erythropoietin in situ and ex vivo. Ex vivo, the erythropoietin may be provided in a cell, tissue or organ preservation solution. For either aspect, the exposing may be by way of continuous perfusion, pulsatile perfusion, infusion, bathing, injection, or catheterization.
Such adverse conditions to function or viability mentioned above may result from clamping of the vascular supply to an organ during repair or other surgical procedure; isolating a viscus or limb during perfusion to provide high levels of, for example, a chemotherapeutic agent; bypass surgery; transcutaneous procedures in which blood flow to a particular tissue or organ may be temporally compromised, such as endarterectomy, angioplasty, stent placement, and cardiac catheterization. Need for protection of cells and tissues also occurs after traumatic amputation or severance of a bodily part such as a finger, toe or limb, such that may be applied to the severed part during the period before reattachment is performed. Administration of the pharmaceutical composition for the beneficial purposes described herein may be provided prior to the period of time during which the adverse conditions prevail, during the period of time, after the period of time, or any combination of times.
An erythropoietin useful for the practice of the invention includes human or another mammalian erythropoietin, an erythropoietin analog, an erythropoietin mimetic, an erythropoietin fragment, a hybrid erythropoietin molecule, an erythropoietin receptor-binding molecule, an erythropoietin agonist, a renal erythropoietin, a brain erythropoietin, an oligomer of any of the foregoing, a multimer of any of the foregoing, a mutein of any of the foregoing, a congener of any of the foregoing, a naturally-occurring form of any of the foregoing, a synthetic form of any of the foregoing, a recombinant form of any of the foregoing, a glycosylation variant of any of the foregoing, a deglycosylated variant of any of the foregoing, or a combination of any of the foregoing. By way of non-limiting example, the amount of an erythropoietin may be within the range from about 100 picograms/ml to about 1,000 nanograms/ml of erythropoietin, an erythropoietin receptor activity modulator, an erythropoietin-activated receptor modulator, or a combination of any of the foregoing. The amount of erythropoietin also may be about 100 nanograms to about 50 micrograms of an erythropoietin per kilogram of the cell, tissue, organ or bodily part. In terms of vascular concentration, the amount of an erythropoietin may be a dose to achieve about 10 picograms/ml to about 1000 nanograms/ml. However, these amounts are merely illustrative and non-limiting.
In one embodiment, the erythropoietin is non-erythropoietic in vivo, such as but not limited to an asialoerythropoietin, such as human asialoerythropoietin. By way of non-limiting example, the amount of an erythropoietin may be within the range from about 100 picograms/ml to about 1,000 nanograms/ml of erythropoietin, an erythropoietin receptor activity modulator, an erythropoietin-activated receptor modulator, or a combination of any of the foregoing. The amount of a non-erythropoietic erythropoietin also may be about 100 nanograms to about 50 micrograms of an erythropoietin per kilogram of the cell, tissue, organ or bodily part. In terms of vascular concentration, the amount of an erythropoietin may be a dose to achieve about 10 picograms/ml to about 1000 nanograms/ml. However, these doses and amounts are merely illustrative and non-limiting.
The foregoing methods are preferably applicable to human beings, but is useful for any mammal, such as but not limited to companion animals, domesticated animals, livestock and zoo animals.
In another aspect of the invention, a method is provided for protecting, maintaining or enhancing the viability or function of a cell, tissue or organ in a mammal, the cell, tissue or organ including at least an erythropoietin-responsive cell or tissue. The cell, tissue or organ is separated from the vasculature of the mammal by an endothelial cell barrier. The method involves at least administering to the vasculature an amount of an erythropoietin for a duration effective to translocate the erythropoietin across the endothelial cell barrier and to protect, enhance or maintain the viability or function of the cell, tissue or organ. By way of non-limiting examples, the administering of the erythropoietin may be provided by a route such as oral, intravenous, topical, intraluminal, inhalation or parenteral administration, the latter including intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, submucosal or intradermal.
By way of non-limiting example, the erythropoietin-responsive cell or tissue may be neuronal, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelial, testes, ovary, or endometrial cells or tissue. These examples of erythropoietin-responsive cells are merely illustrative. In a particular embodiment, the mammalian cell, tissue or organ has expended or will expend a period of time under at least one condition adverse to the viability of the cell, tissue or organ. Such conditions may include traumatic in-situ hypoxia or metabolic dysfunction, surgically-induced in-situ hypoxia or metabolic dysfunction, or in-situ toxin exposure, the latter such as may be associated with chemotherapy or radiation therapy.
An erythropoietin may be dosed to the human being or non-human mammal using an amount, duration and frequency of dosing which achieves the desired enhancement or protection. For chronic administration, it is desirable to avoid increasing the hematocrit by administering an erythropoietin at a dose, duration and frequency of dosing that avoids the erythropoietic activity of the erythropoietin but achieves the desired enhancement as described hereinthroughout. As will be described herein, based on the potency, half-life, and other factors, one of skill in the art may design a dosing regimen which achieves this desired goal.
In the foregoing aspect, the erythropoietin may be human or another mammalian erythropoietin, an erythropoietin analog, an erythropoietin mimetic, an erythropoietin fragment, a hybrid erythropoietin molecule, an erythropoietin receptor-binding molecule, an erythropoietin agonist, a renal erythropoietin, a brain erythropoietin, an oligomer of any of the foregoing, a multimer of any of the foregoing, a mutein of any of the foregoing, a congener of any of the foregoing, a naturally-occurring form of any of the foregoing, a synthetic form of any of the foregoing, a recombinant form of any of the foregoing, a glycosylation variant of any of the foregoing, a deglycosylated variant of any of the foregoing, or a combination of any of the foregoing. In this aspect of the invention, the amount of an erythropoietin may be within the range from about 100 nanograms per kilogram to about 50 micrograms per kilogram of erythropoietin, an erythropoietin receptor activity modulator, an erythropoietin-activated receptor modulator, or a combination thereof, and a pharmaceutically acceptable carrier. A preferred amount of an erythropoietic erythropoietin is about 20 micrograms per kilogram to about 50 micrograms per kilogram. The amount of erythropoietin may also be a dose effective amount to achieve a circulating level of erythropoietin of greater than about 0.5 nanograms/ml to about 1000 nanograms/ml of serum. In a preferred embodiment, the erythropoietin is non-erythropoietic, such as but not limited to an asialoerythropoietin, such as human asialoerythropoietin. In the case of the non-erythropoietic erythropoietin, a preferred amount is 100 nanograms per kilogram up to about 50 micrograms per kilogram; in serum or solution, a concentration of about 10 picograms per milliliter to about 1000 nanograms per milliliter is desirable. Such doses and amounts are merely illustrative and are not intended to be limiting.
In one embodiment, an erythropoietin may have a biological half-life less than 50% that of native human erythropoietin, preferably less than 90% that of native human erythropoietin. As mentioned above, the dose, dosing frequency and duration may be adjusted, particularly for chronic administration, to expose the human being or non-human mammal to an erythropoietin which achieves the desired enhancement or protection but does not induce erythropoiesis or increase hematocrit. Thus, both erythropoietic and non-erythropoietic erythropoietins may be dosed to achieve these desired results. Preferably, for chronic administration, a non-erythropoietic erythropoietin is used. Dosing parameters may also be adjusted for the potency of the particular form of erythropoietin, and particularly a non-erythropoietic form of erythropoietin, for the uses herein.
The foregoing methods are preferably applicable to human beings, but is useful for any mammal, such as but not limited to companion animals, domesticated animals, livestock and zoo animals.
The invention is also directed to a pharmaceutical composition in dosage unit form adapted for modulation of erythropoietin-responsive cells, tissues or organs which includes at least, per dosage unit, an effective non-toxic amount within the range from about 1.5 mg to about 5 mg of an erythropoietin, an erythropoietin receptor activity modulator, an erythropoietin-activated receptor modulator, or a combination of any of the foregoing, and a pharmaceutically acceptable carrier. In one embodiment, the effective non-toxic amount of EPO in the pharmaceutical composition comprises 1 to 5 mg of EPO; 1.5 to 5 mg of EPO; 2 to 5 mg of EPO; 2.5 to 5 mg of EPO; 3.5 to 5 mg of EPO; 4 mg to 5 mg of EPO; or 4.5 to 5 mg of EPO. The erythropoietin may be human or other mammalian erythropoietin, an erythropoietin analog, an erythropoietin mimetic, an erythropoietin fragment, a hybrid erythropoietin molecule, an erythropoietin receptor-binding molecule, an erythropoietin agonist, a renal erythropoietin, a brain erythropoietin, an oligomer of any of the foregoing, a multimer of any of the foregoing, a mutein of any of the foregoing, a congener of any of the foregoing, a naturally-occurring form of any of the foregoing, a synthetic form of any of the foregoing, or a recombinant form of any of the foregoing.
In another aspect, the invention is directed to a pharmaceutical composition in dosage unit form adapted for modulation of erythropoietin-responsive cells, tissues or organs which comprises, per dosage unit, an effective non-toxic amount within the range from about 5 micrograms to 5 mg of a non-erythropoietic erythropoietin, a non-erythropoietic erythropoietin receptor activity modulator, a non-erythropoietic erythropoietin-activated receptor modulator, or a combination of any of the foregoing, and a pharmaceutically acceptable carrier. In a one embodiment, the non-erythropoietic erythropoietin has altered pharmacokinetics compared with native, human erythropoietin, such as but not limited to non-erythropoietic glycosylation variants of erythropoietin or non-erythropoietic deglycosylated variants of erythropoietin; one example being asialoerythropoietin. The ranges may include from about 5 micrograms to 5 mg of a non-erythropoietic erythropoietin; 10 micrograms to 5 mg of a non-erythropoietic erythropoietin; 50 micrograms to 5 mg of a non-erythropoietic erythropoietin; 100 micrograms to 5 mg of a non-erythropoietic erythropoietin; 500 micrograms to 5 mg of a non-erythropoietic erythropoietin; 0.5 mg to 5 mg of a non-erythropoietic erythropoietin; 0.6 mg to 5 mg of a non-erythropoietic erythropoietin; 0.7 mg to 5 mg of a non-erythropoietic erythropoietin; 0.8 mg to 5 mg of a non-erythropoietic erythropoietin; 0.9 mg to 5 mg of a non-erythropoietic erythropoietin; 1 mg to 5 mg of a non-erythropoietic erythropoietin; 1.5 to 5 mg of a non-erythropoietic erythropoietin; 2 to 5 mg of a non-erythropoietic erythropoietin; 2.5 to 5 mg of a non-erythropoietic erythropoietin; 3.5 to 5 mg of a non-erythropoietic erythropoietin; 4 mg to 5 mg of a non-erythropoietic erythropoietin; or 4.5 to 5 mg of a non-erythropoietic erythropoietin.
The invention is also directed to a perfusate solution comprising an erythropoietin, such as but not limited to an erythropoietin, an erythropoietin receptor activity modulator, an erythropoietin-activated receptor modulator, or a combination of any of the foregoing, and a pharmaceutically acceptable carrier. The erythropoietin may be human or another mammalian erythropoietin, an erythropoietin analog, an erythropoietin mimetic, an erythropoietin fragment, a hybrid erythropoietin molecule, an erythropoietin receptor-binding molecule, an erythropoietin agonist, a renal erythropoietin, a brain erythropoietin, an oligomer of any of the foregoing, a multimer of any of the foregoing, a mutein of any of the foregoing, a congener of any of the foregoing, a naturally-occurring form of any of the foregoing, a synthetic form of any of the foregoing, or a recombinant form of any of the foregoing. The perfusate may comprise an erythropoietin at a concentration of about 10 picograms per ml to about 1000 nanograms per ml. However, these amounts are merely illustrative and non-limiting.